The family of steroid and thyroid hormones has become one of the most intensely investigated group of ligands because of their nuclear localization and demonstratable impact on gene transcription. The vitamin D derived secosteroid hormone, 1,25(OH)2 D3, is a member of this family and regulates a multitude of cellular functions in various target tissues. The transcription of the calcium binding protein gene (CaBP-28k) in the intestine demonstrates an apparent absolute requirement for 1,25(OH)2 D3. Like the other steroid hormones, 1,25(OH)2 D3 action is mediated through a high affinity protein receptor that putatively binds to specific DNA domains and in turn participates in the regulation of gene activity. The major emphasis of this proposal is the demonstration of 1,25(OH)2 D3 receptor interaction with vitamin D regulatory elements (DREs) located within the CaBP-28k gene, and that this interaction is integral to the transcriptional regulation of this gene. The DNA upstream of the CaBP gene's first exon should contain the putative regulatory domains. This DNA will be subcloned into plasmid vectors containing reporter genes such as chloramphenicol acetyl transferase (CAT) or human growth hormone (hGH) and transfected into cell lines that express the 1,25 (OH)2 D3 receptor (ROS 17.2.8 3T6, SL-29) and those that do not (ROS 24/1). These experiments will demonstrate if a particular DNA fragment responds to 1,25(OH)2 D3 and requires the receptor to promote and/or enhance the transcription of the reporter gene. Specific receptor-DNA interaction will be established by utilizing filter binding, DNase footprinting and Southwestern-blotting methodology. Once the vitamin D receptor binding domain has been identified, it can be modified by point mutations, switch in orientation, or relocated in order to demonstrate binding specificity and enhancer function. In addition to the vitamin D receptor, various other factors may be involved in transcriptional regulation of the CaBP gene. This proposal is also aimed at beginning to elucidate the tissue specific and developmental regulatory constraints placed on the CaBP gene. Elucidating the mechanism(s) involved in transcriptional regulation of the CaBP gene may lend insight toward understanding how other genes are regulated and how controlling the specific expression of a gene may be accomplished and potentially manipulated in an effort to be utilized as a therapeutic tool.